Effect of soaking on anti-nutritional factors in the sun-dried seeds of hybrid pigeon pea to enhance their nutrients bioavailability

Transcription

1 2018; 7(2): E-ISSN: P-ISSN: JPP 2018; 7(2): Received: Accepted: Reena Devi Department of Biochemistry, CCS Haryana Agricultural University, Hisar, Haryana, India Charul Chaudhary Centre of Food Science and Technology, CCS Haryana Agricultural University, Hisar, Haryana, India Veena Jain Department of Biochemistry, CCS Haryana Agricultural University, Hisar, Haryana, India AK Saxena Department of Biochemistry, Punjab Agricultural University, Ludhiana, Punjab, India Shilpa Chawla Research Scholar, CCS HAU, Hisar, Haryana, India Correspondence Charul Chaudhary Centre of Food Science and Technology, CCS Haryana Agricultural University, Hisar, Haryana, India Effect of soaking on anti-nutritional factors in the sun-dried seeds of hybrid pigeon pea to enhance their nutrients bioavailability Reena Devi, Charul Chaudhary, Veena Jain, AK Saxena and Shilpa Chawla Abstract Certain anti-nutritional factors (ANFs) interact with proteins and form insoluble complexes hence, decreases bioavailability of proteins. Twenty four different hybrid varieties of pigeon pea [Cajanus cajan (L.) Millsp.] were analysed for reduction in ANFs on different soaking treatments. Soaking in distilled water and different salt solutions viz. 1% NaHCO3, mixed salt solutions (MSS) (1.5% NaHCO3+0.5% Na2CO3+0.75% citric acid) led to a significant (P<0.05) reduction in total phenols, orthodihydroxyphenols, tannins and phytates. Phenols and tannins are water soluble and present in seed coat hence, a large reduction observed after simple soaking in distilled water i.e 33.07% and 40.9% respectively. Soaking in distilled water, 1% NaHCO3 and MSS reduced ortho-dihydroxyphenols by 17.39%, 34.78% and 47.83% whereas phytate by 14.9%, 18.3%, and 21% respectively. Amongst various methods studied, soaking in MSS found to be the most effective in reducing levels of ANFs in pigeon pea hybrids. Keywords: pigeon pea, hybrids, anti-nutritional factors (ANFs), soaking, salt solutions, MSS Introduction Pulses constitute one of the richest sources of including valuable but incompletely balance protein, particularly in vegetarian s diet (Ghadge et al., 2008) [20] and are consequently an important part of the people s diet in many parts of the world (Arinathan et al., 2003; 2009) [4, 5]. Among the pulses crops, pigeon pea [Cajanus cajan (L.) millsp.] is the most widely produced and consumed food legume worldwide belongs to family Leguminosae, also known as red gram, arhar, tur dhal. The fruit of the pigeon pea is classified as a pod and each pod contains 3 to 5 seeds which are round or lens in shape. Pigeon pea originated from India and Asia where it travelled to African countries (Onyebuashi, 1986) [33]. India known as the largest producer and consumer of pigeon pea worldwide due to its predominantly grown and consumption. In pigeon pea production, India has highest production of MT in the world followed by Myanmar ( MT), Malawi ( MT), United Republic of Tanzania ( MT), Kenya (89390 MT), Uganda (84200 MT), Dominican Republic (27998 MT) and Nepal (14082 MT) (FAOSTAT, 2012) [19]. The India s 1 st ranks in pulse production have become constant since history of long many back decades (FAOSTAT, 2012) [19]. Pigeon pea is economically and nutritionally important legume as major source of proteins in poor communities of many tropical and subtropical regions of the world viz. India, Myanmar, Malawi, United Republic of Tanzania, Kenya, Uganda, Dominican Republic, Nepal etc. (Singh et al., 1984; ICRISAT 1986; FAOSTAT, 2012) [19, 22]. Inadequate availability and consumption of protein foods in developing countries are a major concern as large segments of population of these countries suffer from protein malnutrition (Arinathan et al., 2009; Soris and Mohan, 2011) [5, 41]. It has been estimated that the total production of legumes provide almost as much protein (20-30%) to the world as wheat and over 50 % more than rice or corn (Rockland et al., 1981; Gopalan et al., 1985) [21, 37]. Nutritionists look for available substitute of protein, which satisfy nutrient requirements of pigeon pea seeds used in daily diet, because of protein possess immense health-related benefits and also contribute the best alternative due to their high nutritive value. They contain a high level of crude protein ranges from 21-30% (Udedibie and Igwe, 1989; Amaefule and Onwudike, 2000) [43, 3]. The composition of matured and dry pigeon pea supply significant amount of energy through water (11.5%), carbohydrates (63.4 %), lipids (1.2 %), dietary fibres (4.4%) and minerals (3.5%), starch also the major constituents of pigeon pea (Ihekoronye and Ngoddy, 1985). In addition, pigeon pea contains reasonable levels of thiamine, riboflavin and niacin (Bressani et al., 1974; Arora, 1977) [8, 6]. In spite of the nutritional potential of the pigeon pea, they are underutilized as food. ~ 675 ~

2 On the other hand, pigeon pea contain some anti-nutritional factors (ANFs) in their seeds and meal which decrease the availability of these nutrients (Ahmed et al., 2006) [2] and may have adverse effects on human nutrition. These ANFs are mainly tannins, phenols, phytic acids, phytates and trypsin inhibitors. It was reported that those ANFs might reduce the nutritional quality of dry beans even if they are present in low concentrations (Abd El-Hady and Habiba, 2003) [1]. Therefore, pigeon peas need to be nutritionally improved and remove or reduce the amount of these compounds. Several treatments such as fermentation, germination, thermal treatments (boiling, cookingand autoclaving),dehulling and soaking procedures have been applied to remove or reduce the amount of these compounds from pigeon peas (Abd El-Hady and Habiba, 2003; Martin-Cabrejas et al., 2004) [1, 27]. In general, these processing techniques improve not only the flavour and palatability of legumes but also increase the protein digestibility, bioavailability of nutrients, by destroying the ANFs (Chau and Cheung, 1997; Martin-Cabrejas et al., 2004; Khandelwal et al., 2010) [27, 9, 25]. As a matter of fact, there is very few published research found on the content and alterations of ANFs of hybrid pigeon pea varieties. The present investigation was carried out with an attempt to gain an insight into the effect of soaking on anti-nutritional factors (ANFs) (viz. phenols, ortho-dihydroxyphenols, tannins and phytates) of pigeon pea [Cajanuscajan (L.) millsp.] to enhance their nutrients bioavailability with the removal of the anti-nutritional factors (ANFs)and make it edible safe protein sources for global consumption. Materials and Methods Seed Samples The laboratory experiments were conducted in the Laboratories of the Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana (Punjab) , India. For this purpose, twenty four hybrids pigeon pea genotypes viz. PHP-23, PHP-24, PHP-25, PHP-26, PHP- 27, PHP-28, PHP-30, PHP-32, PHP-33, PHP-34, PHP-35, PHP-36, PHP-37, PHP-38, PHP-39, PHP-40, PHP-41, PHP- 42, PHP-44, PHP-45, PHP-47, PHP-48, PHP-49, and PHP-50 were procured from Department of Plant Breeding and Genetics of the university. The samples were sun-dried, cleaned to free them from extraneous matter and ground in powder form. The powdered samples were stored in air tight bottles at room temperature (25 C). Sample Preparation (Soaking) The sample preparation depicted in Figure-1. The soaking of seeds of each genotype was done separately in distilled water, 1% sodium bicarbonate solutions and mixed salt solution (MSS) (1.5% NaHCO 3+0.5% Na 2CO % citric acid) for 18 hours. After soaking, the excess of water in the seeds was drained off. The seeds were then dried in an oven at 55 0 C and dried sample was finely ground into flour to pass through a 50 mesh screen for further analysis. Determination of Total Phenol Content Total phenols were estimated using Folin Ciocalteau reagent (Swain and Hillis 1959) [42]. Methanol extract (0.5 ml) was evaporated to dryness and the residue was redissolved in distilled water and mixed thoroughly. To this 0.5 ml of Folin Ciocalteau reagent was added and shaken thoroughly. After 5 min, 1 ml of saturated sodium carbonate solution was added and rested for 1 hour. Absorbance of the blue colour was recorded at 725 nm. Concentration of total phenols was ~ 676 ~ determined from the standard curve which was prepared using μg gallic acid. Determination of Tannins Content Tannins were determined by Folin Denis reagent (Swain and Hillis 1959) [42]. An aliquot of 1 ml was taken and the volume was made to 8.5 ml with distilled water. To this, 0.5 ml of FolinDenis reagent was added. After 3 min, 1 ml of saturated sodium carbonate solution was added. Mixed well and absorbance was measured at 760 nm. Standard curve was prepared using different concentrations of tannic acid. Determination of Ortho-dihydroxy Phenols Content Ortho-dihydroxyphenols were assessed by the method of Nair and Vaidyanathan (1964) [31]. Methanol extract (5 ml) evaporated to dryness. The residue left behind was dissolved in 1 ml of distilled water and shook well. Added 0.5 ml of 10% TCA, 1 ml of 10% sodium tungstate, 0.5 ml of 0.5 N HCl and 1 ml of 0.5 % sodium nitrite and mixed thoroughly. A yellow colour developed. After 5 min, 2 ml of 0.5 N NaOH was added. The colour changes to light cherry colour which was read at 540 nm. Standard curve was prepared using the range of μg of catechol. Extraction and Determination of Phytic Acid Content Extraction for phytic acid was done by refluxing 0.5 g sample with 20 ml of 0.5M nitric acid for 3-4 hrs with continuous shaking followed by centrifugation. The supernatent was used for the estimation. Phytic acid was analysed by the method of Davies and Reid (1971) [11] ml of the filterate was diluted with distilled water to a final volume of 1.4 ml to which 1.0 ml of a solution of ammonium ferric sulphate containing 50 µg iron was added. After thorough mixing, were placed in boiling water bath for 20 min. When cooled to room temperature, 5 ml amyl alcohol and 0.1 ml ammonium thiocyanate solution was added to each test tube. The contents of the test tubes were immediately mixed by inversion and shaking. After centrifugation for a short time (5 min) at low speed, the intensity of the color in the amyl layer was determined at 465 nm against an amyl alcohol blank exactly 15 min after addition of the ammonium thiocyanate solution. Statistical Analysis Experiments were conducted in triplicates. Data were analyzed on the basis of statistical analysis. The mean, standard error, critical difference and their statistical significance were ascertained with the help of software CPCS1 and GSTAT. Results and Discussion The nutritional importance of a given feedstuff in a diet is well evident, depends not only on the nutrient composition, but also on the presence of anti-nutritional factors (ANFs). Hence, elimination or inactivation of ANFs is absolutely necessary to improve the nutritional quality of pigeon pea and effectively utilize their full potential as human food. Total phenolic, tannins, ortho-dihydroxy phenol and phytate content of pigeon pea hybrids is depicted in Table-1. Soaking of pigeon pea seeds is the most simple and inexpensive method for bringing reductions in polyphenols, tannins and other water soluble ANFs. The significant (P<0.05) percent reductions in ANFs of pigeon pea following different soaking treatments is observed as shown in Figure-2. Chopra and Sankhala (2004) [10] stated that soaking is effective in significantly reducing ANFs and concomitantly increasing

3 nutrients bioavailability of legumes. During soaking, legumes come in contact with water which causes leaching of water soluble ANFs into water. The phenolic compounds or their oxidized products form complexes with essential amino acids, enzymes and other proteins, thus lowering their protein digestibility and nutritional values (Shahidi and Naczk 1992) [38]. Total phenol content varied widely from 3.09 mg/g to mg/g with a mean value of 6.56 mg/g in pigeon pea hybrids as given in Table-1. PHP-35 had the lowest phenol content among all hybrids tested followed by PHP-28 and PHP-26. Hence, these genotypes are promising with respect to phenolic content. Figure-3 shows that mean phenolics content decreased from 6.56 mg/g to 4.39 mg/g (percent reduction of 33.07%) by soaking in distilled water. More or less the same percentage losses of phenolics have been observed earlier during soaking in C. cajan (Igbedioh et al., 1995) [23]. Reductions in total phenolic content were 42.8% after soaking in 1% NaHCO 3 (Figure-2). The contents of phenolics in pigeon pea were reduced to the maximum extent when soaked in salt water as compared to distilled water. This accords with an earlier report in Dolichos lablab var. vulgaris (Vijayakumari et al., 1995) [44]. The marked loss of phenolics when subjected to soaking in salt solution may be attributed to its effect in creating an ionic environment. The changed ionic environment in turn might change the seed coat permeability. This enables greater and rapid solid losses (Deshpande, 2002, Mulimani and Supriya, 1994) [8]. The most pronounced effect amongst the salt solutions was registered by soaking in MSS which reduced mean phenolicsupto 2.56 mg/g (60.9% reduction). Significant differences (P<0.5) were observed within processed pigeon pea seed samples. Soaking in salt solution showed an improvement to the protein quality (DeLeon et al., 1992) [12]. The tannin causes a decrease in the digestibility of protein and carbohydrates as a result of the formation of insoluble enzyme-resistant complexes (Reddy et al., 1985). Deshpande et al. (1986) [13] reported that polyphenols could react with proteins and enzymes so they could also act as trypsin inhibitors and amylase inhibitors. Tannin content was ranged between 0.57 to 2.87 mg/g in pigeon pea hybrids. PHP-38 had the lowest tannin content among all hybrids. This accords with an earlier study of Ene-Obong (1995) in pigeon pea but the levels of tannin of pigeon pea hybrids in the present study (Table-1) seem to be slightly higher as compared with that observed by Onweluzoand Nwabugwu (2009). This variation in results may be due to varietal differences. Tannins are located in seed coat and being water soluble, simple soaking can reduce large content of tannins (Figure-2 and 4). Reduction in tannin content was 40.9% due to soaking in distilled water. This was slight similar to an earlier investigation in different varieties of Pisum sativum (Bishnoi et al., 1994) and Viciafaba (Sharma and Sehgal, 1992) [39]. Reduction was not much pronounced after soaking in NaHCO 3 and MSS over soaking in distilled water on the tannin content of the seed samples. Ortho-dihydroxy phenol content varied from 0.11 mg/g to 0.45 mg/g and PHP-38 was found to have the lowest content among all hybrids tested, followed by PHP-47 and PHP-45. Figure-5 shows a very small amount of ortho-dihydroxy phenols was found in hybrids. Soaking in distilled water reduced total orthodihydroxy phenol content upto 17.39%. Percent reduction further increased to 34.78% by soaking in 1% NaHCO 3 and 47.83% by soaking in MSS (Figure-2). Among all the ANFs, phytic acid is one of prime concern for human nutrition and health management (Kumar et al., 2010). The phytate molecule is negatively charged at physiological ph and is reported to bind nutritionally important essential divalent cations, such as iron, zinc, magnesium and calcium. This forms insoluble complexes, thereby making minerals unavailable for absorption (Rimbach et al., 1994). Phytic acid content was exhibited in range of 6.24 mg/g to mg/g (mean 8.62 mg/g). PHP-33, PHP-34 and PHP-35 had the lower content of phytic acid, hence, promising with respect to phytate content. Egli et al. (2002) and Mulimani et al (2003) [29] reported higher values of phytate in millet and pigeon pea. However the disparity may be expected since according to Reddy et al. (1982), phytate level varies in legumes and cereals with variety, cultivar type and soil type among other factors. Effect of soaking on phytate content in pigeon pea hybrids is shown in Figure-6. Phytate is water-soluble, thus a considerable amount of phytate is removed into the water on soaking (14.9%). In addition, this process also enhances the action of naturally occurring phytase in legumes (Kumar et al., 2010). There was 18.3% reduction after soaking in 1% NaHCO 3 and 21% reduction after soaking in MSS for phytic acid (Figure-2). Similar results for reduction in phytic acid in soaked legumes have been reported earlier by Deshpande and Cheryan (1983) [14]. El-Hady and Habiba (2003) [1] observed a 36% reduction in phytic acid in kidney beans after an overnight soaking in water at room temperature. The loss in phytates during soaking of the tested samples may be due to leaching of phytate ions into the soaking water under the influence of a concentration of gradient (difference in chemical potential), which governs the rate of diffusion. Table 1: Anti-nutritional factors (ANFs) content (mg/g) in Pigeon pea [Cajanus cajan (L.) Millsp.] hybrids S. N. Pigeon pea Hybrids Total Phenol Tannin Orthodihydroxy Phenol Phytate 1. PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ± PHP ± ± ± ±0.01 MEAN CD(P<0.05) Values are mean of three replicates ± Standard error ~ 677 ~

4 Fig 1: Flow diagram for sample preparation Fig 2: Percent reduction in ANFs of pigeon pea hybrids by different soaking treatments. Data are expressed as mean ± SE of twenty four pigeon pea hybrids. Significant reductions were observed (P 0.05). ODP, ortho-dihydroxyphenol Fig 4: Effect of different soaking treatment on tannin content (mg/g) in pigeon pea hybrids. Significant differences were observed within processed pigeon pea seed samples w.r.t. total tannin content (P 0.05) Fig 3: Effect of different soaking treatment on phenolics content (mg/g) in pigeon pea hybrids. Significant differences were observed within processed pigeon pea seed samples w.r.t. total phenolics content (P 0.05) Fig 5: Effect of different soaking treatment on orthodihydroxyphenol content (mg/g) in pigeon pea hybrids. Significant differences were observed within processed pigeon pea seed samples w.r.t. ortho-dihydroxyphenol content (P 0.05). ~ 678 ~

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